The collection and storage of immotile sperm has been widely studied in sea urchin and avian species. Despite such efforts, storage of viable samples has been limited to short timeframes, e.g. 6-12 hours with turkey sperm. Indeed, extensive efforts have been directed to developing liquid storage procedures for holding turkey sperm for 24 hours and longer (Bakst, et al., Oviductal Sperm Selection, Transport and Storage in Poultry, POULT. SCI. REV., 5:117-43 (1994); Thurston, Storage of Poultry Semen above Freezing for 24-48 Hours, PROC. FIRST INT'L SYMP. ARTIF. INSEM., 107-22 (1995); Bakst, et al., Modification ofEthidium Bromide Exclusion Procedure for Evaluation of Turkey Sperm, POULT. SCI., 70: 336-70 (1991); Lake, Recent Progress in Poultry Reproduction, WORLD'S POULT. SCI. J., 45:53-59 (1989); Wishart, Physiological Changes in Fowl and Turkey Sperinatozoa During in vitro Storage, B R. POULT. SCI., 30:443-454 (1989); Sexton, Comparison of Commercial Diluents for Holding, TurkTey Sperm 24 Hours at 5.degree. C., POULT. SCI., 67:131-34 (1988); Sexton, Research Note: Influence of Damaged Spermatozoa on the Fertility of Turkey Semen Stored for 24 Hours at 5.degree. C.) However, these studies demonstrate that fertility is generally lower when hens are inseminated with semen stored more than 6 hours.
The basic procedures for semen collection and artificial insemination (AI) for poultry was established in the 1930's (Lake, Historical Perspective of Artificial Insemination Technology, FIRST INT'L. SYMP. ARTIF. INSEM. POULT., pp. 1-20 (1995)). Various methods such as refrigeration, cryopreservation, diluents/buffer systems, detergents, antioxidants and exogenous metabolic inhibitors have been utilized to increase storage viability; however, none of these have proved to be unequivocally successful in obtaining the desired effects.
A significant feature of the reproductive physiology of poultry, for example, is the hen's ability to store fertile sperm for long periods of time. Sperm storage tubules, which are structures found in the distal half of the oviduct of all avian species, sequester and store sperm which are slowly released over time to insure an adequate population of sperm at the site of fertilization (Donoghue, A. M., et al., Storage of Poultry Sperm, 1997, unpublished; Bakst, Oviductal Sperm Storage in Poultry: A Review, REPROD. FERTIL. DEV., 5:595-599 (1993)). Turkey hens inseminated before the onset of egg production can produce fertilized eggs up to 16 weeks after insemination (Christensen et al., Efficacy of Fertilization in Artificially Inseminated Turkey Hens, POULT. SCI., 68:724-729 (1989)). The mechanisms of prolonged sperm storage in the sperm storage tubules are unknown, but they are believed to include reversible suppression of sperm respiration and motility as well as stabilization of the plasma membrane and maintenance of the acrosome (Bakst 1993, supra).
There are many diluents in the prior art to extend and maintain sperm viability in vitro. For example, with turkey semen, diluents such as Beltsville Poultry Semen Extender II, Instruments for Veterinary Medicine formula and Minnesota Turkey Growers Association formula have long been used commercially. The basic characteristics common to nearly all diluents include agents to maintain pH, osmolarity and to provide an energy source for sperm (see review, Christensen, Diluents, Dilution and Storage of Poultry Semen for Six Hours, FIRST INT'L. SYMP. ARTIF. INSEM. POULT., pp. 90-106 (1995)). The buffering agents have included mixtures of phosphates, citrates and/or organic zwitterionic molecules such as BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid) and TES (N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid). One phenomenon apparently consistent with the diluents of the prior art is the decline in fertility with poultry semen stored greater than 6 hours after the first 5 to 8 weeks of egg production. Over the course of egg production, the efficiency of the sperm storage tubules decreases and, thus, even late season declines in fertility and hatch are not uncommon with fresh inseminations. When semen is stored 24 hours or longer, in vitro fertility problems are magnified (Donoghue, et al., 1997, supra). Recently, appropriate concentrations of lipid and amphipathic-soluble antioxidants maintained viability, membrane integrity, and motility of turkey sperm after 48 hour in vitro storage better than the control (Donoghue, et al., Effects of Water- and Lipid-Soluble Antioxidants on Turkey Sperm Viability, Membrane Integrity, and Motility During Liquid Storage, POULT. SCI., 76:1440-45 (1997)). However, maintenance of fertility was not established in vivo. Accordingly, there is a definite need to create a diluent/buffering system which mimics the environment for sperm storage within the hen thereby allowing the in vitro storage of semen for extended periods of time without decreasing in vivo fertility.
Additionally, an improved sperm storage system is needed for sea urchin sperm and sperm of other species. With sea urchins, many studies aimed at examining the intracellular signaling pathways that initiate the activation of flagellar motility have relied on detergent-permeabilized sperm reactivated with exogenous .sup.32 P-ATP (Bracho et al., METHODS CELL BIOL. 47:447-458 (1995); Brokaw, J. CELL. BIOCHEM. 35:175-184 (1987)). However, the normal conditions of sperm collection and reactivation allow variable levels of motility to be expressed prior to analysis (Brokaw, ANN. N.Y. ACAD. SCI. 438:132-141 (1984), as well as phosphorylation of many sperm proteins that are not clearly related to flagellar motility (Chaudhry, et al., CELL MOTIL. CYTOSKELETON 32:65-79 (1995); Tash et al., BIOL. REPROD. 28:75-104 (1983)). Thus, identification of the few relevant proteins that are rapidly phosphorylated during the initial stages of sperm activation is difficult. Metabolic inhibitors or extended incubations at cold temperatures prior to reactivation of motility have been used to try and reverse the effects of background motility. See, e.g., Ahmad et al., ARCH. ANDROL. 35:187-208 (1995); Tash, et al., J. CELL BIOL. 103:649-655 (1986); Lindemann, CELL 13:9-18 (1978). However, these methods depend on endogenous enzyme activities that may reverse the signals originally induced in the background motility. Thus, a reliable method of collecting large amounts of immotile sperm capable of normal activation when required would solve this problem.